1. Field of the Invention
The present invention is related to vascularization inhibitors and a method for producing protein which is useful as a vascularization inhibitor.
2. Description of the Related Art
It is known in clinical field that, when a carcinoma is removed, other small tumors which were in a state of lull become activated to start growing or to raise their metastatic inclination. As a reason for this phenomena, the carcinoma itself is considered to produce some substances, which would inhibit the growth of other tumors. Angiostatin is known as one of such substances.
O'Reilley, et al. (Cell. Vol. 79, 315-328, Oct. 21, 1994, J. Folkman's Research Group) showed that urine and serum from mice having tumors inhibited metastasis of carcinoma in mice as well as growth of endothelial cells participating in vascularization. The substance showing such an inhibitory effect was purified and identified as a protein of 38 kDa by analysis of SDS-PAGE. This protein showed at least 98% homology to an internal fragment of 38 kDa plasminogen which fragment has N-terminal portion starting at mino acid 98 of plasminogen. (See the Discussion on the left column of page 323 of the above-mentioned reference.)
This protein called "angiostatin" has been found to coincides with a partial sequence of plasminogen. The plasminogen is a protein in plasma having 5 domains called "kringles". The kringle is a domain of triple loops formed by disulfide bonds. The domain is considered to play some role in linkages in which membranes, proteins and phospholipids are involved, and in regulating the proteolytic activity of enzymes. Hereinafter the five kringles will be referred to as "K1" to "K5" starting with K1 close to the N-terminus along the plasminogen molecule.
The structure of human plasminogen is shown in FIGS. 1-3. FIG. 1 is a partial modification of the figure on page 276 of "Hemostasis, Thrombus, Fibrinogenolysis", (Edited by Michio Matsuda, et al., p.276, First Edition published in 1994, Chugai-Igakusya Co. Tokyo, Japan). Plasminogen is a protein consisting of 791 amino acids and contains 5 kringles K1-K5. The amino acid sequence of plasminogen has been determined (FIG. 1). FIG. 1 shows this amino acid sequence, using one letter code. In FIG. 1, "A" means alanine, "C" means cysteine, "D" means asparagine acid, "E" means glutamic acid, "F" means phenylalanine, "G" means glycine, "H" means histidine, "I" means isoleucine, "K" means lysine, "L" means leucine, "M" means methionine, "N" means asparagine, "P" means proline, "Q" means glutamine, "R" means arginine, "S" means serine, "T" means threonine, "V" means valine, "W" means tryptophane, and "Y" means tyrosine. Human plasminogen has four an elastase hydrolytic sites. The peptide bonds at amino acids between position 78 and position 79, position 338 and position 339, position 354 and position 355, and position 440 and position 441 (or position 442 and position 443) can be hydrolyzed with an elastase. (These bonds are indicated by {character pullout} in FIG. 1 and .tangle-solidup. in FIGS. 2 and 3.)
Glycosylation sites exist at 289 and 346 amino acids of plasminogen (as shown by .diamond-solid. in FIG. 1, .circle-solid. in FIG. 2 and .smallcircle. in FIG. 3). Plasminogen has two kinds of isoforms. One is attached to a sugar chain at amino acid 289 (Form 1), and the other has no sugar chain at amino acid 289 (Form 2).
Plasminogen has a lysine-binding site 1 in K1-K3 and another lysine-binding site 2 in K4 (marked by .star. in FIG. 3), and so it can bind with lysine at these sites. In purifying plasminogen from plasma, Lysine-Sepharose chromatography can be used because of its property of binding with lysine. K5 has an aminohexal-binding site (marked by .circleincircle. in FIG. 3).
The hydrolysis of human plasminogen with an elastase by O'Reilly, et al. was carried out under conditions so as to hydrolyze the peptide bonds at amino acids between positions 78 and 79, positions 338 and 339, positions 354 and 355, and positions 440 and 441 (or positions 442 and 443). Therefore, the resultant fragments are those from amino acids 1-78 (N-terminal potion of plasminogen), amino acids 79-338 (fragments containing K1-K3 considered to be a mixture of those derived from plasminogen in Form 1 and those derived from that in Form 2), amino acids 339-354 (a short fragment between K3 and K4), amino acids 355-440 (a fragment containing K4), and amino acids 441(or 443)-791 (the portion containing K5 and C-terminal portion of plasminogen, called "miniplasminogen").
Using the fragment containing K1-K3, the fragment containing K4 and miniplasminogen, O'Reilly, et al. carried out some experiments for the inhibitory effect on the growth of endothelial cells and on the metastasis of carcinoma. As the result, the inhibitory effects on the growth of endothelial cells and metastasis of carcinoma was observed only in the fragments containing K1-K3. On the contrary, no effect was observed either in the fragment containing K4 only or in human plasminogen itself.
O'Reilly, et al. named the fragments containing K1-K3 "purified angiostatin". Purified angiostatin is considered to inhibit the growth and metastasis of carcinoma cells by preventing vascularization.
Angiostatin can control the metastatic inclination of carcinoma, which would otherwise extremely increase after removal of the carcinoma, to the same level as before the carcinoma removal. In other words, the metastasis of carcinoma is inhibited after its removal by administering the same amount of angiostatin as produced by the carcinoma itself. Furthermore, angiostatin is obtained as a result of hydrolysis of plasminogen, which existing abundantly in the form of protein in plasma, as a substrate with an elastase which is a protease produced by vascular endothelial cells. As angiostatin itself is a protein existing in blood vessels, it is digested by a protease like plasmin. Therefore, it is considered that angiostatin can be used as an anticancer agent naturally occurring in a living body with almost no side effect. In addition, plasminogen is abundantly present in plasma, it is convenient to use plasminogen as a starting raw material for the production and purification of angiostatin. These suggest that angiostatin is excellent as one of known anti-metastatic agents.
In addition to angiostatin, fumagillin is also known as a vascularization inhibitor. Fumagillin is a kind of antibiotics produced by microorganisms that is different from angiostatin. Japanese Patent Publication No. 6-60095 discloses that fumagillin and its derivatives can be used as a vascularization inhibitor.
For using angiostatin as a carcinoma metastasis inhibitor, it is necessary to hydrolyze plasminogen extracted from plasma with an elastase and fractionate on columns. This fractionation can be presently effected only by HPLC. If angiostatin is to be extracted and purified according to such procedures, not only it is time-consuming and costly but also it shows low recovery. Besides, it is difficult to store angiostatin because of its strong inclination to decompose.
It is problematic to use fumagillin in clinical aspects because of its strong adverse side effects, though it is also effective like angiostatin in inhibiting vascularization and metastasis of carcinoma.
Moreover, it is disclosed in BBRC, Vol. 102, 1, 46-52, 1981 and Chemical Reviews, Vol. 81, Oct. 5, 1981 that the fragment consisting of amino acids 355-791 was obtained by limiting hydrolysis with an elastase. It is stated in the references that, after separating plasminogen into Form 1 and Form 2, the fragment bound on the Lysine-Sepharose column is eluted at gradient concentrations of -aminocaproic acid (hereinafter referred to as "EACA"), and that the several eluted peaks are further fractionated by gel filtration chromatography. However, the use of the gradient concentration of EACA and gel filtration chromatography as mentioned in the references is not suitable for recovering intermediate products like the fragment consisting of amino acids 355-791 of plasminogen, because these techniques need a longer time. Moreover, according to the references, the fragment consisting of the amino acids 355-791 is dealt with simply as an activated plasminogen but with no experiment to show any function of such fragments.
Therefore, there is a demand for a novel substance inhibiting vascularization with little adverse effects and a simple method of purifying angiostatin.